The Tox21 program is a federal collaboration among researchers from NIH, including NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS), the Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA). These agencies work together to advance in vitro toxicological testing. The Tox21 program is comprised of the following specialties: Genomic Toxicology, Systems Toxicology, and Computational Toxicology. The Genomic Toxicology team has developed high-throughput gene expression technologies with the goal of annually generating data from hundreds of thousands of samples across a vast number of human genes. RNA-seq technology was optimized and robustly automated in the unique solid-state 384-well plate format. Experiments successfully multiplexed 1,041 assays representing 347 cellular stress genes in each of 384 samples, which were in turn pooled/multiplexed for a single NextGen sequencing reaction comprising approximately 200 million sequences. A thorough evaluation of the NCATS RASL-Seq platform showed excellent reproducibility (average 10-11% CV), high-throughput (samples: 6 x 384-well plates in one week), 90% concordance with qPCR, and moderate cost (<$12/sample). The RASL-seq platform was used to analyze dose-responses for 61 mitochondrial membrane modulators, 19 tobacco components and 19 antiviral nucleoside analog drugs. Data analyses were scripted into a series of automated steps, or pipeline for both RASL-seq data analyses and RNA-seq whole-genome gene expression data analyses established. The Tox21 Genomic Toxicology team also has improved cellular models for hepatocytes, neurons and vascular endothelial cells. Tox21 Phase II includes detailed analysis of compounds that are found to be active in primary quantitative high-throughput screening. This secondary screening will require improved cellular models, typically immortalized cells, or stem cells, which are differentiated in vitro into functional hepatocytes, neurons and cardiomyocytes. For neuronal toxicity, the team compared SH-SY5Y neuroblastoma cells, LUHMES conditionally immortalized dopaminergic human neurons and neuronal stem cells as models of neuronal toxicity. They found that LUHMES cells are most sensitive to most of these compounds, and most surprising, that neurons differentiated from LUHMES cells were more sensitive to 11 toxicants that the parent undifferentiated cell line. Lastly, the Tox21 Genomic Toxicology team has used induced pluripotent stem cell (iPSC) technology to generate disease-in-a-dish models. For example, in the tobacco project, both RASL-seq and RNA-seq were used to help the team understand the effects of tobacco components on vascular endothelial cells. By producing these cells using iPSC from smokers with and without vascular disease, the team was able to determine whether some patients are genetically susceptible or resistant to the disease. The Tox21 Genomic Toxicology team discovered that iPSC-derived endothelial cells (iECs) in fact responded to smoke very similarly to primary endothelial cells from three vascular beds, validating the iEC model. They repeated an RNA-seq experiment that establishes that genetic/epigenetic differences between four individuals govern distinct responses to tobacco smoke. This experiment utilized 12 iPSC-derived endothelial cell lines from the four individuals and found that the three cell lines from each individual were very reproducible. They also assessed the dose-dependent cytotoxicity and gene expression responses to 19 selected tobacco components on human umbilical vein endothelial cells as well as normal iECs.